RESUMO
Viruses have developed sophisticated strategies to control metabolic activity of infected cells in order to supply replication machinery with energy and metabolites. Dengue virus (DENV), a mosquito-borne flavivirus responsible for dengue fever, is no exception. Previous reports have documented DENV interactions with metabolic pathways and shown in particular that glycolysis is increased in DENV-infected cells. However, underlying molecular mechanisms are still poorly characterized and dependence of DENV on this pathway has not been investigated in details yet. Here, we identified an interaction between the non-structural protein 3 (NS3) of DENV and glucokinase regulator protein (GCKR), a host protein that inhibits the liver-specific hexokinase GCK. NS3 expression was found to increase glucose consumption and lactate secretion in hepatic cell line expressing GCK. Interestingly, we observed that GCKR interaction with GCK decreases DENV replication, indicating the dependence of DENV to GCK activity and supporting the role of NS3 as an inhibitor of GCKR function. Accordingly, in the same cells, DENV replication both induces and depends on glycolysis. By targeting NAD(H) biosynthesis with the antimetabolite 6-Amino-Nicotinamide (6-AN), we decreased cellular glycolytic activity and inhibited DENV replication in hepatic cells. Infection of primary organotypic liver cultures (OLiC) from hamsters was also inhibited by 6-AN. Altogether, our results show that DENV has evolved strategies to control glycolysis in the liver, which could account for hepatic dysfunctions associated to infection. Besides, our findings suggest that lowering intracellular availability of NAD(H) could be a valuable therapeutic strategy to control glycolysis and inhibit DENV replication in the liver.
Assuntos
Vírus da Dengue , Dengue , Glucoquinase , Glicólise , NAD , Proteínas não Estruturais Virais , Replicação Viral , Glicólise/efeitos dos fármacos , Vírus da Dengue/efeitos dos fármacos , Glucoquinase/metabolismo , Glucoquinase/antagonistas & inibidores , Humanos , Replicação Viral/efeitos dos fármacos , Proteínas não Estruturais Virais/metabolismo , Proteínas não Estruturais Virais/genética , Animais , Dengue/tratamento farmacológico , Dengue/virologia , Dengue/metabolismo , NAD/metabolismo , NAD/biossíntese , Linhagem Celular , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Glucose/metabolismo , Fígado/virologia , Fígado/metabolismo , Antivirais/farmacologia , Proteases Virais , Serina Endopeptidases , Nucleosídeo-Trifosfatase , RNA Helicases DEAD-boxRESUMO
Chagas disease is one of the world's neglected tropical diseases, caused by the human pathogenic protozoan parasite Trypanosoma cruzi. There is currently a lack of effective and tolerable clinically available therapeutics to treat this life-threatening illness and the discovery of modern alternative options is an urgent matter. T. cruzi glucokinase (TcGlcK) is a potential drug target because its product, d-glucose-6-phosphate, serves as a key metabolite in the pentose phosphate pathway, glycolysis, and gluconeogenesis. In 2019, we identified a novel cluster of TcGlcK inhibitors that also exhibited anti-T. cruzi efficacy called the 3-nitro-2-phenyl-2H-chromene analogues. This was achieved by performing a target-based high-throughput screening (HTS) campaign of 13,040 compounds. The selection criteria were based on first determining which compounds strongly inhibited TcGlcK in a primary screen, followed by establishing on-target confirmed hits from a confirmatory assay. Compounds that exhibited notable in vitro trypanocidal activity over the T. cruzi infective form (trypomastigotes and intracellular amastigotes) co-cultured in NIH-3T3 mammalian host cells, as well as having revealed low NIH-3T3 cytotoxicity, were further considered. Compounds GLK2-003 and GLK2-004 were determined to inhibit TcGlcK quite well with IC50 values of 6.1 µM and 4.8 µM, respectively. Illuminated by these findings, we herein screened a small compound library consisting of thirteen commercially available 3-nitro-2-phenyl-2H-chromene analogues, two of which were GLK2-003 and GLK2-004 (compounds 1 and 9, respectively). Twelve of these compounds had a one-point change from the chemical structure of GLK2-003. The analogues were run through a similar primary screening and confirmatory assay protocol to our previous HTS campaign. Subsequently, three in vitro biological assays were performed where compounds were screened against (a) T. cruzi (Tulahuen strain) infective form co-cultured within NIH-3T3 cells, (b) T. brucei brucei (427 strain) bloodstream form, and (c) NIH-3T3 host cells alone. We report on the TcGlcK inhibitor constant determinations, mode of enzyme inhibition, in vitro antitrypanosomal IC50 determinations, and an assessment of structure-activity relationships. Our results reveal that the 3-nitro-2-phenyl-2H-chromene scaffold holds promise and can be further optimized for both Chagas disease and human African trypanosomiasis early-stage drug discovery research.
Assuntos
Benzopiranos , Glucoquinase , Tripanossomicidas , Trypanosoma cruzi , Animais , Humanos , Camundongos , Benzopiranos/farmacologia , Benzopiranos/química , Doença de Chagas/tratamento farmacológico , Doença de Chagas/parasitologia , Descoberta de Drogas/métodos , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/química , Glucoquinase/metabolismo , Glucoquinase/antagonistas & inibidores , Ensaios de Triagem em Larga Escala , Simulação de Acoplamento Molecular , Células NIH 3T3 , Relação Estrutura-Atividade , Tripanossomicidas/farmacologia , Tripanossomicidas/química , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/enzimologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologiaRESUMO
Eighteen amino sugar analogues were screened against Trypanosoma cruzi glucokinase (TcGlcK), a potential drug-target of the protozoan parasite in order to assess for viable enzyme inhibition. The analogues were divided into three amino sugar scaffolds that included d-glucosamine (d-GlcN), d-mannosamine (d-ManN), and d-galactosamine (d-GalN); moreover, all but one of these compounds were novel. TcGlcK is an important metabolic enzyme that has a role in producing G6P for glycolysis and the pentose phosphate pathway (PPP). The inhibition of these pathways via glucose kinases (i.e., glucokinase and hexokinase) appears to be a strategic approach for drug discovery. Glucose kinases phosphorylate d-glucose with co-substrate ATP to yield G6P and the formed G6P enters both pathways for catabolism. The compound screen revealed five on-target confirmed inhibitors that were all from the d-GlcN series, such as compounds 1, 2, 4, 5, and 6. Four of these compounds were strong TcGlcK inhibitors (1, 2, 4, and 6) since they were found to have micromolar inhibitory constant (Ki) values around 20 µM. Three of the on-target confirmed inhibitors (1, 5, and 6) revealed notable in vitro anti-T. cruzi activity with IC50 values being less than 50 µM. Compound 1 was benzoyl glucosamine (BENZ-GlcN), a known TcGlcK inhibitor that was the starting point for the design of the compounds in this study; in addition, TcGlcK - compound 1 inhibition properties were previously determined [D'Antonio, E. L. et al. (2015) Mol. Biochem. Parasitol. 204, 64-76]. As such, compounds 5 and 6 were further evaluated biochemically, where formal Ki values were determined as well as their mode of TcGlcK inhibition. The Ki values determined for compounds 5 and 6 were 107 ± 4 µM and 15.2 ± 3.3 µM, respectively, and both of these compounds exhibited the competitive inhibition mode.
Assuntos
Amino Açúcares/farmacologia , Inibidores Enzimáticos/farmacologia , Glucoquinase/antagonistas & inibidores , Trypanosoma cruzi/enzimologia , Amino Açúcares/síntese química , Amino Açúcares/química , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Glucoquinase/metabolismo , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
Glucokinase (GLK) and Hexokinase (HK) have been characterized as essential targets in Trypanosoma cruzi (Tc)-mediated infection. A recent study reported the propensity of the concomitant inhibition of TcGLK and TcHK by compounds GLK2-003 and GLK2-004, thereby presenting an efficient approach in Chagas disease treatment. We investigated this possibility using atomic and molecular scaling methods. Sequence alignment of TcGLK and TcHK revealed that both proteins shared approximately 33.3 % homology in their glucose/inhibitor binding sites. The total binding free energies of GLK2-003 and GLK2-004 were favorable in both proteins. PRO92 and THR185 were pivotal to the binding and stabilization of the ligands in TcGLK, likewise their conserved counterparts, PRO163 and THR237 in TcHK. Both compounds also induced a similar pattern of perturbations in both TcGLK and TcHK secondary structure. Findings from this study therefore provide insights into the underlying mechanisms of dual inhibition exhibited by the compounds. These results can pave way to discover and optimize novel dual Tc inhibitors with favorable pharmacokinetics properties eventuating in the mitigation of Chagas disease.
Assuntos
Inibidores Enzimáticos/farmacologia , Glucoquinase/antagonistas & inibidores , Hexoquinase/antagonistas & inibidores , Tripanossomicidas/farmacologia , Trypanosoma cruzi/enzimologia , Doença de Chagas/tratamento farmacológico , Doença de Chagas/parasitologia , Inibidores Enzimáticos/química , Glucoquinase/química , Glucoquinase/metabolismo , Hexoquinase/química , Hexoquinase/metabolismo , Humanos , Modelos Moleculares , Termodinâmica , Tripanossomicidas/química , Trypanosoma cruzi/efeitos dos fármacosRESUMO
Maturity-onset diabetes of the young (MODY) type 2 is caused by heterozygous inactivating mutations in the gene encoding glucokinase (GCK), a pivotal enzyme for glucose homeostasis. In the pancreas GCK regulates insulin secretion, while in the liver it promotes glucose utilization and storage. We showed that silencing the Drosophila GCK orthologs Hex-A and Hex-C results in a MODY-2-like hyperglycemia. Targeted knock-down revealed that Hex-A is expressed in insulin producing cells (IPCs) whereas Hex-C is specifically expressed in the fat body. We showed that Hex-A is essential for insulin secretion and it is required for Hex-C expression. Reduced levels of either Hex-A or Hex-C resulted in chromosome aberrations (CABs), together with an increased production of advanced glycation end-products (AGEs) and reactive oxygen species (ROS). This result suggests that CABs, in GCK depleted cells, are likely due to hyperglycemia, which produces oxidative stress through AGE metabolism. In agreement with this hypothesis, treating GCK-depleted larvae with the antioxidant vitamin B6 rescued CABs, whereas the treatment with a B6 inhibitor enhanced genomic instability. Although MODY-2 rarely produces complications, our data revealed the possibility that MODY-2 impacts genome integrity.
Assuntos
Diabetes Mellitus Tipo 2/genética , Instabilidade Genômica/genética , Glucoquinase/genética , Estresse Oxidativo/genética , Animais , Glicemia/genética , Diabetes Mellitus Tipo 2/patologia , Modelos Animais de Doenças , Drosophila/genética , Drosophila/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento/genética , Glucoquinase/antagonistas & inibidores , Produtos Finais de Glicação Avançada/genética , Heterozigoto , Humanos , Hiperglicemia/genética , Hiperglicemia/patologia , Larva/genética , Larva/crescimento & desenvolvimento , Mutação/genética , Vitamina B 6/metabolismoRESUMO
This paper presents the synthesis and glucokinase activity of novel hydrazone derivatives. The 2-(4-cyclopropylsulfonylphenyl)-2-[(E)-pyrrolidin-1-ylimino]-acetamide derivatives 5a-5h presented the in vitro glucokinase activities and in vivo blood glucose-lowering effects in mice. Particularly, 5h showed an oral hypoglycemic effect in rats at 1â¯mg/kg. These hydrazone derivatives are a potential new class of glucokinase activators for the treatment of type 2 diabetes.
Assuntos
Acetamidas/farmacologia , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Tipo 2/tratamento farmacológico , Glucoquinase/antagonistas & inibidores , Hipoglicemiantes/farmacologia , Tiazóis/farmacologia , Acetamidas/administração & dosagem , Acetamidas/síntese química , Administração Oral , Animais , Glicemia/efeitos dos fármacos , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Relação Dose-Resposta a Droga , Desenho de Fármacos , Glucoquinase/metabolismo , Hipoglicemiantes/administração & dosagem , Hipoglicemiantes/síntese química , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , Ratos , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Tiazóis/administração & dosagem , Tiazóis/síntese químicaRESUMO
Berberine (BBR) is a widely used anti-diabetic agent, and liver glucokinase (GK) has been reported to be involved. However, the mechanisms of BBR in regulating GK are still unknown. Here, we found that BBR upregulated GK immunofluorescence expression in AML12 cells cultured in high glucose and increased glycogen content simultaneously. BBR improved hyperglycemia in db/db mice, and increased liver glucose-6-phosphate/glucose-1-phosphate (G-6-P/G-1-P) was found by analyzing metabolites (serum, liver, and feces) based on gas chromatography-mass spectrometry (GC-MS) metabolomics. Pharmacokinetics-pharmacodynamics (PK-PD) assessment revealed enriched BBR distribution in the liver, and liver G-6-P had the same trend as the concentration-time curve of BBR. G-6-P is solely catalyzed by GK, and GK activity and expression showed a positive correlation with liver BBR levels. In db/db mice, BBR also upregulated GK in liver fractions (cytoplasm and nucleus) and liver glycogen content. GK functionally worked by dissociating from GK regulatory protein (GKRP), and although GKRP expression was not affected, we found a decreased ratio of GK binding with GKRP in BBR treated db/db mice. In conclusion, our study suggests the dissociation of GK from GKRP as the potential mechanism for liver GK increase upon BBR treatment, which contributes to the anti-diabetic effect of BBR.
Assuntos
Berberina/farmacologia , Diabetes Mellitus Tipo 2/tratamento farmacológico , Glucoquinase/antagonistas & inibidores , Hiperglicemia/tratamento farmacológico , Animais , Glicemia/efeitos dos fármacos , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Glucoquinase/genética , Glucose/metabolismo , Hepatócitos/efeitos dos fármacos , Humanos , Hiperglicemia/genética , Hiperglicemia/patologia , Hipoglicemiantes/farmacologia , Insulina/genética , Fígado/efeitos dos fármacos , Fígado/enzimologia , Glicogênio Hepático/efeitos adversos , Masculino , Camundongos , Camundongos Endogâmicos NODRESUMO
The aim was to characterize a phenolic-rich water extract from the pericarp of an improved genotype of Apache red maize (RPE) and evaluate its ability to activate the type 2 diabetes markers free fatty acid receptor 1 (GPR40) and glucokinase (GK) in vitro. The extract contained mainly phenolic acids, anthocyanins, and other flavonoids. RPE inhibited α-amylase (IC50 = 88.3 µg/mL), α-glucosidase (IC50 = 169.3 µg/mL), and reduced glucose transport in a Caco-2 cell monolayer (up to 25%). Furthermore, RPE activated GPR40 (EC50 = 77.7 µg/mL) in pancreatic INS-1E cells and GK (EC50 = 43.4 µg/mL) in liver HepG2 cells, potentially through allosteric modulation. RPE activated GPR40-related insulin secretory pathway and activated the glucose metabolism regulator AMPK (up to 78%). Our results support the hypothesis that foods with a high concentration of anthocyanins and phenolic acids, such as in the selected variety of maize used, could ameliorate obesity and type 2 diabetes comorbidities.
Assuntos
Antocianinas/farmacologia , Diabetes Mellitus Tipo 2/metabolismo , Flavonoides/farmacologia , Glucoquinase/antagonistas & inibidores , Hidroxibenzoatos/farmacologia , Extratos Vegetais/farmacologia , Receptores Acoplados a Proteínas G/metabolismo , Zea mays/química , Antocianinas/química , Antocianinas/isolamento & purificação , Transporte Biológico/efeitos dos fármacos , Células CACO-2 , Diabetes Mellitus Tipo 2/genética , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Flavonoides/química , Flavonoides/isolamento & purificação , Glucoquinase/metabolismo , Glucose/metabolismo , Células Hep G2 , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Hidroxibenzoatos/química , Hidroxibenzoatos/isolamento & purificação , Insulina/metabolismo , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Fígado/citologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Simulação de Acoplamento Molecular , Pâncreas/citologia , Pâncreas/efeitos dos fármacos , Pâncreas/metabolismo , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Receptores Acoplados a Proteínas G/genética , Zea mays/genética , alfa-Amilases/antagonistas & inibidores , alfa-Amilases/metabolismoRESUMO
Glucose metabolism in humans is tightly controlled by the activity of glucokinase (GCK). GCK is predominantly produced in the pancreas, where it catalyzes the rate-limiting step of insulin secretion, and in the liver, where it participates in glycogen synthesis. A multitude of disease-causing mutations within the gck gene have been identified. Activating mutations manifest themselves in the clinic as congenital hyperinsulinism, while loss-of-function mutations produce several diabetic conditions. Indeed, pharmaceutical companies have shown great interest in developing GCK-associated treatments for diabetic patients. Due to its essential role in maintaining whole-body glucose homeostasis, GCK activity is extensively regulated at multiple levels. GCK possesses a unique ability to self-regulate its own activity via slow conformational dynamics, which allows for a cooperative response to glucose. GCK is also subject to a number of protein-protein interactions and post-translational modification events that produce a broad range of physiological consequences. While significant advances in our understanding of these individual regulatory mechanisms have been recently achieved, how these strategies are integrated and coordinated within the cell is less clear. This review serves to synthesize the relevant findings and offer insights into the connections between molecular and cellular control of GCK.
Assuntos
Glucoquinase/metabolismo , Animais , Proteínas de Transporte/fisiologia , Ativação Enzimática , Glucoquinase/antagonistas & inibidores , Glucoquinase/química , Glucose/análise , Humanos , Fosfofrutoquinase-2/metabolismo , Ligação Proteica , Conformação Proteica , Processamento de Proteína Pós-Traducional , Proteína SUMO-1/metabolismoRESUMO
An early sign of islet failure in type 2 diabetes (T2D) is the loss of normal patterns of pulsatile insulin release. Disruptions in pulsatility are associated with a left shift in glucose sensing that can cause excessive insulin release in low glucose (relative hyperinsulinemia, a hallmark of early T2D) and ß-cell exhaustion, leading to inadequate insulin release during hyperglycemia. Our hypothesis was that reducing excessive glucokinase activity in diabetic islets would improve their function. Isolated mouse islets were exposed to glucose and varying concentrations of the glucokinase inhibitor d-mannoheptulose (MH) to examine changes in intracellular calcium ([Ca2+]i) and insulin secretion. Acutely exposing islets from control CD-1 mice to MH in high glucose (20 mM) dose dependently reduced the size of [Ca2+]i oscillations detected by fura-2 acetoxymethyl. Glucokinase activation in low glucose (3 mM) had the opposite effect. We then treated islets from male and female db/db mice (age, 4 to 8 weeks) and heterozygous controls overnight with 0 to 10 mM MH to determine that 1 mM MH produced optimal oscillations. We then used 1 mM MH overnight to measure [Ca2+]i and insulin simultaneously in db/db islets. MH restored oscillations and increased insulin secretion. Insulin secretion rates correlated with MH-induced increases in amplitude of [Ca2+]i oscillations (R2 = 0.57, P < 0.01, n = 10) but not with mean [Ca2+]i levels in islets (R2 = 0.05, not significant). Our findings show that correcting glucose sensing can restore proper pulsatility to diabetic islets and improved pulsatility correlates with enhanced insulin secretion.
Assuntos
Cálcio/metabolismo , Glucoquinase/antagonistas & inibidores , Secreção de Insulina/efeitos dos fármacos , Ilhotas Pancreáticas/efeitos dos fármacos , Manoeptulose/farmacologia , Animais , Diabetes Mellitus Tipo 2/metabolismo , Modelos Animais de Doenças , Feminino , Glucoquinase/metabolismo , Glucose , Insulina/metabolismo , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Masculino , Camundongos , Camundongos EndogâmicosRESUMO
In higher eukaryotes, several ATP-utilizing enzymes known as hexokinases activate glucose in the glycolysis pathway by phosphorylation to glucose 6-phosphate. In contrast to canonical hexokinases, which use ATP, ADP-dependent glucokinase (ADPGK) catalyzes noncanonical phosphorylation of glucose to glucose 6-phosphate using ADP as a phosphate donor. Initially discovered in Archaea, the human homolog of ADPGK was described only recently. ADPGK's involvement in modified bioenergetics of activated T cells has been postulated, and elevated ADPGK expression has been reported in various cancer tissues. However, the physiological role of ADPGK is still poorly understood, and effective ADPGK inhibitors still await discovery. Here, we show that 8-bromo-substituted adenosine nucleotide inhibits human ADPGK. By solving the crystal structure of archaeal ADPGK in complex with 8-bromoadenosine phosphate (8-Br-AMP) at 1.81 Å resolution, we identified the mechanism of inhibition. We observed that 8-Br-AMP is a competitive inhibitor of ADPGK and that the bromine substitution induces marked structural changes within the protein's active site by engaging crucial catalytic residues. The results obtained using the Jurkat model of activated human T cells suggest its moderate activity in a cellular setting. We propose that our structural insights provide a critical basis for rational development of novel ADPGK inhibitors.
Assuntos
Adenosina/análogos & derivados , Glucoquinase/química , Adenosina/química , Adenosina/farmacologia , Domínio Catalítico , Cristalografia por Raios X , Glucoquinase/antagonistas & inibidores , Glucose/metabolismo , Humanos , Células Jurkat , Conformação ProteicaRESUMO
The glucokinase regulatory protein (GKRP) plays an essential role in glucose homeostasis by acting as a competitive inhibitor of glucokinase (GCK) and triggering its localization to the hepatocyte nucleus upon glucose deprivation. Metabolites such as fructose 6-phosphate and sorbitol 6-phosphate promote assembly of the GCK-GKRP complex, whereas fructose 1-phosphate and functionalized piperazines with potent in vivo antidiabetic activity disrupt the complex. Here, we establish the molecular basis by which these natural and synthetic ligands modulate the GCK-GKRP interaction. We demonstrate that a small-molecule disruptor of the protein-protein interaction utilizes a two-step conformational selection mechanism to associate with a rare GKRP conformation constituting 3% of the total population. Conformational heterogeneity of GKRP is localized to the N-terminus and deleting this region eliminates the ability of sorbitol 6-phosphate to promote the GCK-GKRP interaction. Stabilizing ligands favor an extended N-terminus, which sterically positions two arginine residues for optimal Coulombic interaction with a pair of carboxylate side chains from GCK. Conversely, disruptors promote a more compact N-terminus in which an interfacial arginine residue is stabilized in an unproductive orientation through a cation-π interaction with tyrosine 75. Eliminating the ability to sample this binding impaired conformation enhances the intrinsic inhibitory activity of GKRP. Elucidating the molecular basis of ligand-mediated control over the GCK-GKRP interaction is expected to impact the development and future refinement of therapeutic agents for diabetes and cardiovascular disease, which result from improper GKRP regulation of GCK.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/química , Glucoquinase/antagonistas & inibidores , Hipoglicemiantes/farmacologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Relação Dose-Resposta a Droga , Glucoquinase/metabolismo , Humanos , Hipoglicemiantes/química , Ligantes , Modelos Moleculares , Estrutura Molecular , Ligação Proteica/efeitos dos fármacos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Relação Estrutura-AtividadeRESUMO
AIM: Small molecule activators of glucokinase (GKAs) have been explored extensively as potential anti-hyperglycaemic drugs for type 2 diabetes (T2D). Several GKAs were remarkably effective in lowering blood glucose during early therapy but then lost their glycaemic efficacy chronically during clinical trials. MATERIALS AND METHODS: We used rat hepatocytes to test the hypothesis that GKAs raise hepatocyte glucose 6-phosphate (G6P, the glucokinase product) and down-stream metabolites with consequent repression of the liver glucokinase gene ( Gck). We compared a GKA with metformin, the most widely prescribed drug for T2D. RESULTS: Treatment of hepatocytes with 25 mM glucose raised cell G6P, concomitantly with Gck repression and induction of G6pc (glucose 6-phosphatase) and Pklr (pyruvate kinase). A GKA mimicked high glucose by raising G6P and fructose-2,6-bisphosphate, a regulatory metabolite, causing a left-shift in glucose responsiveness on gene regulation. Fructose, like the GKA, repressed Gck but modestly induced G6pc. 2-Deoxyglucose, which is phosphorylated by glucokinase but not further metabolized caused Gck repression but not G6pc induction, implicating the glucokinase product in Gck repression. Metformin counteracted the effect of high glucose on the elevated G6P and fructose 2,6-bisphosphate and on Gck repression, recruitment of Mlx-ChREBP to the G6pc and Pklr promoters and induction of these genes. CONCLUSIONS: Elevation in hepatocyte G6P and downstream metabolites, with consequent liver Gck repression, is a potential contributing mechanism to the loss of GKA efficacy during chronic therapy. Cell metformin loads within the therapeutic range attenuate the effect of high glucose on G6P and on glucose-regulated gene expression.
Assuntos
Ativadores de Enzimas/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Glucoquinase/metabolismo , Hepatócitos/efeitos dos fármacos , Hipoglicemiantes/farmacologia , Metformina/farmacologia , Tiazóis/farmacologia , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Células Cultivadas , Dieta Ocidental/efeitos adversos , Frutose/administração & dosagem , Frutose/efeitos adversos , Frutosedifosfatos/metabolismo , Glucoquinase/antagonistas & inibidores , Glucoquinase/química , Glucoquinase/genética , Glucose-6-Fosfatase/antagonistas & inibidores , Glucose-6-Fosfatase/química , Glucose-6-Fosfatase/genética , Glucose-6-Fosfatase/metabolismo , Glucose-6-Fosfato/metabolismo , Hepatócitos/citologia , Hepatócitos/metabolismo , Hepatócitos/patologia , Masculino , Camundongos Endogâmicos C3H , Sobrepeso/enzimologia , Sobrepeso/metabolismo , Sobrepeso/patologia , Regiões Promotoras Genéticas/efeitos dos fármacos , Piruvato Quinase/antagonistas & inibidores , Piruvato Quinase/química , Piruvato Quinase/genética , Piruvato Quinase/metabolismo , Ratos WistarRESUMO
Ligand-target residence time is emerging as a key drug discovery parameter because it can reliably predict drug efficacy in vivo. Experimental approaches to binding and unbinding kinetics are nowadays available, but we still lack reliable computational tools for predicting kinetics and residence time. Most attempts have been based on brute-force molecular dynamics (MD) simulations, which are CPU-demanding and not yet particularly accurate. We recently reported a new scaled-MD-based protocol, which showed potential for residence time prediction in drug discovery. Here, we further challenged our procedure's predictive ability by applying our methodology to a series of glucokinase activators that could be useful for treating type 2 diabetes mellitus. We combined scaled MD with experimental kinetics measurements and X-ray crystallography, promptly checking the protocol's reliability by directly comparing computational predictions and experimental measures. The good agreement highlights the potential of our scaled-MD-based approach as an innovative method for computationally estimating and predicting drug residence times.
Assuntos
Glucoquinase/química , Simulação de Dinâmica Molecular , Cristalografia por Raios X , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Glucoquinase/antagonistas & inibidores , Glucoquinase/metabolismo , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/química , Isoenzimas/metabolismo , Cinética , Ligantes , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-Atividade , Fatores de TempoRESUMO
The glucokinase regulatory protein (GKRP) is a competitive inhibitor of glucokinase (GCK), triggering its localization to the hepatocyte nucleus upon glucose deprivation. Here we establish the kinetic mechanism of GKRP action by analyzing its association with a genetically encoded, fluorescent variant of human GCK. Our results demonstrate that binding of GKRP to GCK involves two steps, formation of an initial encounter complex followed by conformational equilibration between two GKRP-GCK states. Fructose 6-phosphate, a known enhancer of GKRP action, promotes formation of the initial encounter complex via a 2.6-fold increase in kon and stabilizes the complex through a 60-fold decrease in koff.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Frutosefosfatos/farmacologia , Glucoquinase/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Glucoquinase/genética , Glucoquinase/metabolismo , Humanos , Cinética , Conformação ProteicaRESUMO
Liver glucose metabolism is dependent on glucokinase activity. Glucokinase expression is transcriptionally regulated by hormones and metabolites of glucose, and glucokinase activity is dependent on reversible binding of glucokinase to a specific inhibitor protein, glucokinase regulatory protein (GKRP), and to other binding proteins such as 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase (PFK2/FBP2), which functions as an activator. Glucokinase is inhibited in the postabsorptive state by sequestration in the nucleus bound to GKRP, and it is activated postprandially by portal hyperglycemia and fructose through dissociation from GKRP, translocation to the cytoplasm, and binding to PFK2/FBP2. Glucagon dissociates this interaction, promoting translocation back to the nucleus. In humans, changes in glucokinase expression and activity are associated with poorly controlled type 2 diabetes and with nonalcoholic fatty liver disease, and a common variant of GKRP with altered binding affinity for glucokinase is associated with increased blood and liver lipids and other metabolic traits that implicate a role for GKRP in maintaining intrahepatic metabolite homeostasis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Regulação Enzimológica da Expressão Gênica , Glucagon/metabolismo , Glucoquinase/metabolismo , Fígado/enzimologia , Modelos Biológicos , Fosfofrutoquinase-2/metabolismo , Animais , Diabetes Mellitus Tipo 2/enzimologia , Diabetes Mellitus Tipo 2/metabolismo , Glucoquinase/antagonistas & inibidores , Glucoquinase/química , Glucoquinase/genética , Homeostase , Humanos , Fígado/metabolismo , Hepatopatia Gordurosa não Alcoólica/enzimologia , Hepatopatia Gordurosa não Alcoólica/metabolismo , Período Pós-Prandial , Transporte ProteicoRESUMO
The oxytocin (OT) and vasopressin (VP) neurons of the supraoptic nucleus (SON) demonstrate characteristics of "metabolic sensors". They express insulin receptors and glucokinase (GK). They respond to an increase in glucose and insulin with an increase in intracellular [Ca(2+)] and increased OT and VP release that is GK dependent. Although this is consistent with the established role of OT as an anorectic agent, how these molecules function relative to the important role of OT during lactation and whether deficits in this metabolic sensor function contribute to obesity remain to be examined. Thus, we evaluated whether insulin and glucose-induced OT and VP secretion from perifused explants of the hypothalamo-neurohypophyseal system are altered during lactation and by diet-induced obesity (DIO). In explants from female day 8 lactating rats, increasing glucose (Glu, 5 mM) did not alter OT or VP release. However, insulin (Ins; 3 ng/ml) increased OT release, and increasing the glucose concentration in the presence of insulin (Ins+Glu) resulted in a sustained elevation in both OT and VP release that was not prevented by alloxan, a GK inhibitor. Explants from male DIO rats also responded to Ins+Glu with an increase in OT and VP regardless of whether obesity had been induced by feeding a high-fat diet (HFD). The HFD-DIO rats had elevated body weight, plasma Ins, Glu, leptin, and triglycerides. These findings suggest that the role of SON neurons as metabolic sensors is diminished during lactation, but not in this animal model of obesity.
Assuntos
Gorduras na Dieta/farmacologia , Lactação/metabolismo , Neurônios/metabolismo , Obesidade/metabolismo , Ocitocina/metabolismo , Núcleo Supraóptico/metabolismo , Vasopressinas/metabolismo , Aloxano/farmacologia , Animais , Desidratação/metabolismo , Dieta Hiperlipídica , Inibidores Enzimáticos/farmacologia , Feminino , Glucoquinase/antagonistas & inibidores , Glucoquinase/metabolismo , Glucose/farmacologia , Técnicas In Vitro , Insulina/farmacologia , Ratos , Ratos Sprague-Dawley , Receptor de Insulina/biossínteseRESUMO
Monogenic diabetes is a rare genetic type of diabetes caused by pancreatic ß-cells dysfunction. All subtypes of monogenic diabetes are recognized in the pediatric population. They include maturity onset diabetes of the young, permanent neonatal diabetes mellitus and rare syndromic forms of diabetes. An early and proper diagnosis allows to implement an optimal treatment, leads to improved metabolic control and amelioration of related disabilities as well as increases the quality of life of the patients.
Assuntos
Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus/genética , Adulto , Síndrome de Alstrom/diagnóstico , Síndrome de Alstrom/genética , Síndrome de Bardet-Biedl/diagnóstico , Síndrome de Bardet-Biedl/genética , Diabetes Mellitus/diagnóstico , Diabetes Mellitus Tipo 2/diagnóstico , Glucoquinase/antagonistas & inibidores , Humanos , Células Secretoras de Insulina/citologia , Mutação , Polônia , Qualidade de Vida , Reino Unido , Síndrome de Wolfram/diagnóstico , Síndrome de Wolfram/genéticaRESUMO
Glucose concentration changes in the nucleus tractus solitarius (NTS) affect visceral function and metabolism by influencing central vagal circuits, especially inhibitory, GABAergic NTS neurons. Acutely elevated glucose can alter NTS neuron activity, and prolonged hyperglycemia and hypoinsulemia in animal models of type 1 diabetes results in plasticity of neural responses in the NTS. NTS neurons contributing to metabolic regulation therefore act as central glucose sensors and are functionally altered in type 1 diabetes. Glucokinase (GCK) mediates cellular utilization of glucose, linking increased glucose concentration to excitability changes mediated by ATP-sensitive K(+) channels (KATP). Using quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), Western blot, and in vitro electrophysiology, we tested the hypothesis that changes in GCK expression in the NTS accompany the development of diabetes symptoms in the streptozotocin (STZ)-treated mouse model of type 1 diabetes. After several days of hyperglycemia in STZ-treated mice, RNA expression of GCK, but not Kir6.2 or SUR1, was decreased versus controls in the dorsal vagal complex. Electrophysiological recordings in vitro indicated that neural responses to acute hyperglycemia, and synaptic responsiveness to blockade of GCK with glucosamine, were attenuated in GABAergic NTS neurons from STZ-treated mice, consistent with reduced molecular and functional expression of GCK in the vagal complex of hyperglycemic, STZ-treated mice. Altered autonomic responses to glucose in type 1 diabetes may therefore involve reduced functional GCK expression in the dorsal vagal complex.
Assuntos
Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/fisiopatologia , Glucoquinase/metabolismo , Núcleo Solitário/metabolismo , Núcleo Solitário/fisiopatologia , Potenciais de Ação/efeitos dos fármacos , Animais , Diabetes Mellitus Tipo 1/induzido quimicamente , Modelos Animais de Doenças , Neurônios GABAérgicos/metabolismo , Glucoquinase/antagonistas & inibidores , Glucosamina/farmacologia , Glucose/farmacologia , Canais KATP/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Estreptozocina , Potenciais Sinápticos/efeitos dos fármacosRESUMO
In the new human EndoC-ßH1 ß-cell line, a detailed analysis of the physiological characteristics was performed. This new human ß-cell line expressed all target structures on the gene and protein level, which are crucial for physiological function and insulin secretion induced by glucose and other secretagogues. Glucose influx measurements revealed an excellent uptake capacity of EndoC-ßH1 ß-cells by the Glut1 and Glut2 glucose transporters. A high expression level of glucokinase enabled efficient glucose phosphorylation, increasing the ATP/ADP ratio along with stimulation of insulin secretion in the physiological glucose concentration range. The EC50 value of glucose for insulin secretion was 10.3 mM. Mannoheptulose, a specific glucokinase inhibitor, blocked glucose-induced insulin secretion (GSIS). The nutrient insulin secretagogues l-leucine and 2-ketoisocaproate also stimulated insulin secretion, with a potentiating effect of l-glutamine. The Kir 6.2 potassium channel blocker glibenclamide and Bay K 8644, an opener of the voltage-sensitive Ca(2+) channel significantly potentiated GSIS. Potentiation of GSIS by IBMX and forskolin went along with a strong stimulation of cAMP generation. In conclusion, the new human EndoC-ßH1 ß-cell line fully mirrors the analogous physiological characteristics of primary mouse, rat and human ß-cells. Thus, this new human EndoC-ßH1 ß-cell line is very well suited for physiological ß-cell studies.